Immunoassay methodology for the diagnostic determination of biological analytes (drugs, enzymes, metabolites, hormones, antigens, etc.) has proven valuable for clinical analyses, primarily because of the highly specific recognition between analytes and antibodies elicited for those analytes. Although extensively used, the cost and time intensiveness of these methods and the safety hazards of radioimmunoassay have prompted investigations of new techniques. Much attention has been given to "biosensors" in which an immunological reaction that occurs at the interface of a transducer results in output of an electrical signal. Critical features of biosensors are low cost, simplicity, disposability, and sensitivity.
Nucleic acid hybridization tests, which make use of specific polynucleotide probes, provide a means of detecting specific sequences of nucleic acids in test samples and thereby provide important new clinical diagnostic capability. For example, susceptibility to a disease as well as the identity of organisms which might be involved can be evaluated. Hybridization tests have established relationships between viral infections and cancer. Prenatal diagnosis of genetic disease and detection of inherited disease traits have been reported. Applications for the identification of slow growing and resistant infectious organisms have been reported. (Skylar, "DNA Hybridization in Diagnostic Pathology", Human Path., 16, 654 (1986)).
Mixed-phase hybridization systems typically have been used to perform these types of tests. In testing, the hybridizations are performed on membranes (solid phase), usually consisting of nylon or nitrocellulose. As such, the tests are quite cumbersome involving complicated multistep procedures. For example, the assays usually involve loading a membrane with a nucleic acid sample by fixing the nucleic acid to the membrane (if DNA, it must be denatured to create single-stranded molecules) and then saturating the remaining membrane attachment sites with heterologous nucleic acids to prevent the probe reagent from sticking to the membrane in a nonspecific manner. All of these steps must be done before performing the actual hybridization with reporter reagents. The conventional membrane based test procedures are time consuming, taking 4-24 hours to perform, burdensome and complex, requiring multiple reagent additions, wash procedures, and labor intensive manipulations. (Gore, Clin. Chem. News, 12, 1 (1986)).
Furthermore, membrane-based hybridizations cannot always be used directly for crude samples. The membranes are subject to clogging. Moreover, crude samples contain proteins, lipids, mucopolysaccharides, etc., which compete for binding sites on the membranes, reduce the binding capacity of the membrane and contribute to nonspecific binding of reporter reagents. These competing interactions cause unacceptable background and diminished test response. Furthermore, the nucleic acid is typically found only in minute quantities (&lt;10.sup.-15 M) in most test samples, since only a few copies of target DNA are present in each cell. Therefore, for clinical diagnostic applications the nucleic acids must be partially purified and concentrated prior to testing.
A number of new hybridization techniques circumventing these drawbacks have been reported in the literature. The sandwich hybridization technology on various supports has reduced sample pretreatment complexity and decreased the number of procedural steps.
Despite simplified sample pretreatment, sandwich assays continue to suffer from long equilibration times, procedural complexity and limited sensitivity. This results from the concentration dependence of the hybridization reactions which dictate that longer equilibration times are required at lower target concentrations (discussed in "Nucleic Acid, Hybridizations.", B. D. Hermes and D. J. Higgins, eds., IRL Press, 1985), and the insensitivity associated with various instruments and procedures for detection of reporter probe reagents.
Sandwich hybridizations require two independent hybridization events. The reactions times are influenced by both the reporter probe and capture reagent concentrations. Furthermore, the reaction rates are known to be slower on solid phase reagents than would occur in solution. It is therefore desirable to have a test method in order to shorten the assay time. A desirable advance would be a test method that permits each hybridization to take place in solution.
Hybridization assays and immunoassays are severely limited for both diagnostic and research applications by the lack of detection sensitivity. Generally, only a few copies of target gene sequences or target analyte are found in samples of clinical interest. For example, clinically important, infectious disease specimens generally contain between 1 and 10.sup.6 infectious organisms. Since each organism contains only a few copies (4 to 100) of a specific sequence of genetic information per cell, the total target DNA available ranges from 10.sup.-15 to 10.sup.-20 moles. This is below the detection limit of many hybridization methods. For this reason, probe tests generally have not been used for direct specimen testing, but have been useful for testing specimens in which the number of microbes has been increased by culturing or replicating the specific gene sequence of interest.
To overcome sensitivity limits, various detection approaches for hybridization assays have been used. One such method relies on radio-labeled reporter reagents, but is widely considered hazardous and impractical. Other detection methods make use of fluorescent tags or enzyme labeling by which fluorescent products are generated. Although these are highly sensitive techniques, detection of fluorescence and luminescence are inherently limited because the intensity of the detection signal is subject to decay from photo bleaching and quenching.
Gene amplification strategies have also been disclosed in the art to increase the sensitivity of probe tests. Gene Probe Inc. described the use of probes directed against RNA target. Since many copies of target RNA can be produced in each cell during the expression of a single copy of DNA, RNA probe tests tend to be inherently more sensitive and thus more useful for direct specimen testing. However, RNA targets are particularly labile and are subject to enzymatic digestion by ribonucleases ubiquitously found in samples. Cetus Corp. (Emeryville, Calif.) has reported the development of an in vitro gene amplification technique using a polymerase enzyme to multiply the number of DNA copies found in test samples. In this way the number of copies of DNA is greatly increased (ca. a million fold). Once expanded, the target DNA can then be tested using conventional probe analysis techniques. Biotechnolgy News, Oct. 16 (1986). This technique, known as a polymerase chain reaction procedure, involves multiple steps adding time, additional user manipulations, and reagent costs to the overall probe assay.
It is therefore desirable to have a method of detecting hybridization assays which is highly sensitive, less complex than known techniques, can be performed safely, and is not subject to interferences from chemical quenching reactions and light absorbing materials. The instant invention seeks to overcome the above mentioned limitations by exploiting a method of enzyme amplified piezoelectric detection of nucleic acid sequences.
The use of a piezoelectric quartz crystal microbalance (QCM) device has been reported for immunoassay applications and detection of polynucleotide such as DNA. This device consists of a single quartz crystal wafer sandwiched between two metal electrodes. The electrodes provide means of connecting the device to an external oscillator circuit that drives the quartz crystal at its resonant frequency. This frequency is dependent on the mass of the crystal, as well as the mass of any layers confined to the electrode areas of the crystal. Changes in mass on the surface of the electrode thus change the frequency of the QCM. The changes in the resonant frequency of these devices can be correlated to the amount of mass change. If the quartz crystal and any attached layers are presumed to obey rigid-layer behavior, the mass change can be determined from the frequency change by the Sauerbrey relationship. ##EQU1## where .DELTA.f is the measured frequency shift, f.sub.0 the parent frequency of the quartz crystal, .DELTA.m the mass change, A the piezoelectrically active area, .rho..sub.q the density of quartz (2.648 g cm.sup.-3) and .mu..sub.q the shear modulus (2.947.times.10.sup.11 dynes cm.sup.-2 for AT-cut quartz).
Applications of the QCMs to immunoassay and hybridization generally involve attaching the first member of a specific binding pair to the surface of the QCM before the actual analysis.
The piezoelectric methods described in the art do not teach means of using enzymes as a means to amplify the piezoelectric detection of polynucleic acids. Detection sensitivity of the art is thus inherently limited by the weight of the specific polynucleic acids or by the increased mass achieved by use of a particle reporter.
Each of these methods involves first determining the resonance frequency of the crystal. A sample suspected of containing the second member of the binding pair is then added under conditions suitable for promoting binding between the two members of the binding pair. The excess sample debris and unbound material is freed from the QCM by washing. Then the crystal is measured prior to or after drying of the crystal.
In EPO 295,965 for example, the mass change is attributed only to the mass increase resulting solely from the binding of the second member of the binding pair to the QCM. Consequently, sensitivity is poor. There is therefore a need in the art for a piezoelectric based hybridization method in which the mass change resulting from the specific binding between the complementary strands can be amplified to provide a more sensitive and reliable assay.
Unlike immunoassays in which the assay conditions can be essentially standardized for different methods and thus more easily automated, hybridization assays require careful consideration of optimum reaction conditions. Complicating the design of a reaction system is the fact that different polynucleotides hybridize under different conditions. For example, denatured DNA in the presence of its complementary strand will hybridize under proper conditions and re-associate into double stranded DNA. The extent of hybridization is related to the degree of complementarity between the two strands, the ionic strength, chain length, polynucleic acid concentrations, temperature and pH of the hybridization media as discussed in "Nucleic Acid, Hybridizations", B. D. Harmes and D. J. Higgins, eds., IRL Press, 1985. As a consequence, optimum hybridization conditions tend to differ for each unique target sequence. The variation in reaction conditions severely complicates the automation of probe tests by requiring means to vary temperature, reaction conditions and timing for each different probe test.
It is therefore desirable to have a piezoelectric nucleic acid hybridization assay that (1) permits hybridization under reaction conditions required for each different hybridization and (2) is performed independent of the conditions for detecting successful hybridizations by the piezoelectric oscillator. This would enable different hybridization assays to be carried out using the same procedure and (or) detected under the same measurement conditions.
A major limitation of the art is that the surface of the piezoelectric crystal must be modified by attaching to it one member of the specific binding pair. As a consequence, each specific test requires a uniquely modified piezoelectric crystal. Receptor reagents are expensive, can be inactivated during the immobilization process and can separate from the solid surface after immobilization. (G. G. Giulbault, J. H. Luong, and E. Pursak-Sochaczewski, Biotechnology, Vol 7, pp 349-351, (1989)). Preparation of the assay reagents can thus be complicated and require unique reaction conditions for each type of analyte. It is therefore of practical advantage to have a sandwich assay system which enables the same immobilized surface capture reagent to be used for all tests regardless of the specific target analyte.
A need also exists for methodology to anchor a surface capture reagent to the electrode surface of the piezoelectric oscillator, thus forming the piezoelectric sensor in the proper format.
It is the purpose of this invention to provide an assay method which is rapid, procedurally uncomplicated and capable of affording both quantitative and qualitative results. The instant invention adopts a sandwich hybridization assay system which enables the same surface capture reagent to be used for all tests regardless of the nucleic acid target. It is also less expensive to use a non-nucleic acid material as a capture reagent on the oscillator.
The instant invention uses a piezoelectric hybridization method which enables the hybridization phases of the assay to be carried out independently of the attachment of surface capture reagents to the piezoelectric oscillator. Therefore, hybridization conditions can be readily varied. Further, the method of this invention permits automation and more effective replication of the tests because the attachment of a target complex to the piezoelectric sensor can be carried out under the same conditions, regardless of the conditions used for hybridization.